Scoparone Inhibits Ultraviolet Radiation-Induced Lipid Peroxidation
Paul Sourivonga, Kristina Schronerova´b, and Mela´nia Babincova´b,*
a Willis-Knighton Cancer Center, Department of Radiation Oncology, 2600 Kings Highway, Shreveport, Louisiana 71103, USA
b Department of Biophysics, Comenius University, Mlynska´ dolina F1, 842 48 Bratislava, Slovakia. Fax: +42 12 65 42 58 82. E-mail: babincova@fmph.uniba.sk
* Author for correspondence and reprint requests
Z. Naturforsch.62 c, 61Ð64 (2007); received August 8/September 7, 2006
Antioxidant capabilities of scoparone, the component ofArtemisia scopariaand other me- dicinal plants, against lipid peroxidation induced by ultraviolet radiation or Fenton reaction have been analyzed. Lipid peroxidation was monitored by measuring the absorption spectra of the conjugated dienes and quantified by the Klein oxidation index. Obtained results imply that scoparone is a very efficient inhibitor of ultraviolet radiation-induced lipid peroxidation and damage.
Key words: Artemisia scoparia, Ultraviolet Radiation, Lipid Peroxidation
Introduction
Acute and chronic exposure to solar radiation is linked to a number of types of skin damage, such as phototoxicity, photoallergy, aggravation of pre- existing skin diseases, distinct photodermatoses, as well as to immunosupression, photosenescence and photocarcinogenesis (Guercio-Hauer et al., 1994). Ultraviolet radiation is subdivided into four groups: UVA radiation (320Ð400 nm), UVB radiation (280Ð320 nm), UVC radiation (200Ð 280 nm), and vacuum UV radiation with wave- lengths less than 200 nm. The effect of UV radia- tion on multicellular organisms is localized in the skin. When a photon reaches the tissue-air bound- ary, part of the beam is reflected. The remaining light enters the tissue, where the absorption can begin. Radiation has to penetrate through thestra- tum corneum before reaching viable tissue, and therefore the thickness and composition of the stratum corneum always represents a modifying factor (Anderson and Parrish, 1981). Having reached viable tissue, the radiation can be absorbed by chromophores (melanin), whose amount varies between individuals. The effect of radiation on the cells mainly depends on its wavelength and on the amount of the radiation (Morliereet al., 1995).
Peroxidation process leads to the damage of membranes and changes the biophysical proper- ties. For example, an increased permeability of the lipid bilayer, decreased fluidity and lowered elec- tric resistance of membranes has been observed as
0939Ð5075/2007/0100Ð0061 $ 06.00 ”2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com ·D
the results of peroxidation. Oxidation process can be initiated by the reactive oxygen species (ROS), such as the superoxide anion OÐ2, hydroxyl radical
·OH or singlet oxygen1O2. Accumulation of ROS in aerobic organisms is thought to cause oxidative damage in cells. Oxidative damage is believed to be strongly associated with certain human patho- logical processes and diseases such as carcinogene- sis, mutagenesis, aging, arthritis, and atherosclero- sis. Peroxidation damage caused by UV radiation in the biological membranes and in the liposomes was confirmed in multiple studies (Ming-Kuei and Miao, 1999). Each organism has its own mecha- nism of protection against the free radicals activity, however, peroxidation can be also suppressed by natural inhibitors called antioxidants (Trommeret al., 2001; Dobarganes and Velasco, 2002).
One of the potential antioxidants is scoparone (6,7-dimethoxy-2H-1-benzopyran-2-one), which belongs to the group of coumarins. More than 300 coumarins have been identified from natural sour- ces, especially green plants. The pharmacological and biochemical properties and therapeutic appli- cations of simple coumarins depend upon the pat- tern of substitution. Scoparone has been isolated from the hypolipidaemic Chinese herb Artemisia scopariaand shown to reduce the proliferative re- sponses of human peripheral mononuclear cells, to relax smooth muscle, to reduce total cholesterol and triglycerides and to retard the characteristic pathomorphological changes in hypercholestero-
62 P. Sourivonget al.· Scoparone and Lipid Peroxidation laemic diabetic rabbits. Various properties of scop-
arone were suggested to account for these find- ings, including the ability to scavenge the reactive oxygen species, to inhibite of tyrosine kinases and to potentiate of prostaglandin generation (Huang et al., 1993; Liu et al., 2001, 2002; Van Peltet al., 1989; Zhaoet al., 2000).
Material and Methods Liposome preparation
Lipid soy-bean phosphatidylcholine (Sigma, St.
Louis, USA) was dissolved in organic solvents (a mixture of chloroform and methanol 2 :1 v/v). The lipid solution was evaporated in vacuo. After evaporation of the solvent, Tris [2-(hydroxy- methyl)-2-amino-1,3-propanediol] buffer (pH 7.4) and scoparone, α-tocopherol and hyaluronic acid (all obtained from Sigma) with desired concentra- tions were added into a glass vessel with lipid film, and the solution was shaken mechanically. The suspension was then sonicated with a Labsonic 2000 sonicator (Braun Biotech, Göttingen, Ger- many) at 80 W for 15 min under nitrogen atmos- phere in an ice-bath in order to obtain a clear sus- pension of liposomes with a phosphatidylcholine concentration of 3.5 mm.
Induction of lipid peroxidation by UV radiation Peroxidative process was initiated by ultraviolet radiation (UVA) with a wavelength range 320Ð 400 nm generated by a 75 W UV lamp (Philips, Hamburg, Germany). Homogenous liposome sus- pension with 2 mm thickness containing varying concentrations of scoparone or another antioxi- dant used (α-tocopherol or hyaluronic acid) was exposed to UVA radiation for 75 min.
Induction of lipid peroxidation by Fenton reaction To generate the hydroxyl radical (·OH) so as to test the antioxidant efficacy of the prepared plant extracts, the Fenton (HaberÐWeiss) reaction was used. FeCl2reacts with hydrogen peroxide in the following manner:
Fe2++ H2O25Fe3++ ·OH + HOÐ.
Fenton reaction was initiated by the addition of H2O2 and FeCl2 with a final concentration of 100 mm and 2 mm, respectively, to the liposome suspension containing varying concentrations of antioxidants.
Determination of oxidation index
Absorption spectra of the conjugated dienes were recorded in the wavelength range 215Ð 320 nm using a UV MINI 1240 UV-VIS spectro- photometer (Shimadzu, Kyoto, Japan). The in- crease of the absorption at 233 nm was considered as an evidence of the formation of the conjugated dienes, and the oxidation index was calculated from the ratio of the absorbance values (A233/ A215) (Klein, 1970; Babincova´, 1994; Babincova´
and Sourivong, 2001; Babincova´et al., 1999, 2002).
Results and Discussion
Peroxidation of lipids is a measure of damage to the membrane lipids caused by the attack of the reactive oxygen species. Inhibition of lipid peroxi- dation by any external agent is often used to eva- luate its antioxidant capacity. Peroxidation of the fatty acids of phospholipids occurs via a free radi- cal chain mechanism. Formation of lipid free radi- cals is initiated by the abstraction of a hydrogen atom from the lipid chain. The most susceptible to degradation are lipids containing double bonds, since unsaturation permits delocalization of the re- maining unpaired electrons along the lipid chain.
Polyunsaturated lipids are thus particularly prone to oxidative degradation. In the presence of oxy- gen, the process further proceeds via the forma- tion of the hydroperoxides, which degrade sponta- neously to form aldehydes with concomitant fission of the fatty acid chain. Natural phospholi- pids contain only non-conjugated double bonds and, therefore, have a UV absorbance peak at a very short wavelength (200Ð205 nm). Removal of a hydrogen atom from the methylene group lo- cated between two double bonds spreads the un- saturation over five carbon atoms and results in the formation of a conjugated diene which is ener- getically more favourable than the two isolated double bonds. As a result, the second absorbance maximum at 233 nm appears.
At first we have studied lipid peroxidation in- duced by UVA radiation. Until not long ago, UVA radiation was considered to be beneficial to the skin, and prevention against acute sunburn mainly concentrated on protection against the harmful ef- fects of exposure to UVB rays (De Gruijl, 2000).
However, upon deeper investigation, it was dis- covered that UVA radiation is most cytotoxic to human skin cells. These rays penetrate deeper into the dermis, where they provoke dermal connective
P. Sourivonget al.· Scoparone and Lipid Peroxidation 63 tissue alterations associated with photoaging and
many other subchronic to chronic skin disorders (Schaeferet al., 2000). In fact, UVA radiation has been shown to induce lipid peroxidation in liposo- mal, micellar, and natural systems (Bose and Chat- terjee, 1995; Boseet al., 1989).
The main role in lipid peroxidation induced by UV radiation is played by singlet oxygen1O2. Sin- glet oxygen can be generated e.g.by the reaction
Fig. 1. Dependence of the Klein oxidation index on the scoparone concentration. Lipid peroxidation was initi- ated with 75 min exposition to ultraviolet radiation. Er- ror bars represent standard deviations of a mean value (n= 10).
Fig. 2. Comparison of antioxidative activities of all used substances (with the following concentrations: 200μm hyaluronic acid, 800μmα-tocopherol, and 120μmscop- arone). Lipid peroxidation was initiated with 75 min ex- position to ultraviolet radiation. Error bars represent standard deviations of a mean value (n= 10).
Fig. 3. Comparison of antioxidative activities of all used substances (with the following concentrations: 200μm hyaluronic acid, 800μmα-tocopherol, and 120μmscop- arone). Lipid peroxidation was initiated by the Fenton reaction. Error bars represent standard deviations of a mean value (n= 10).
of two peroxyl radicals. UVA radiation can also induce the formation of hydroxyl radicals (Halli- well and Gutteridge, 1993).
The dependence of the Klein peroxidation index on scoparone concentration is shown in Fig. 1.
From the dose-response curve of scoparone-scav- enging activities it was found that the scavenging activity increased with the increase of the scopar- one concentration and that the scavenging effect is most pronounced starting from the concentration 120μmfor both UVA- as well as Fenton reaction- induced free radical formation. For a comparison we have also monitored conjugated diene forma- tion in the presence of α-tocopherol, which is known to be incorporated into the lipid bilayer, and in the presence of hyaluronic acid, which is a water-soluble polysaccharide (Figs. 2, 3). It is clear, scoparone is a very potent antioxidant, also effec- tive at very low concentrations, with capabilities even better than that of these two well-known free radical scavengers. We found that scoparone is less efficient when the free radical formation was initi- ated using the Fenton reaction (Fig. 3).
Acknowledgement
This work was supported by the grant VEGA 1/
2012/05 from Slovak grant agency.
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